PROJECT SUMMARY Preterm birth occurs in an estimated 10% of pregnancies, causing high rates of morbidity and mortality in infants. This public health problem has led to domestic and international initiatives including the US Surgeon General's Conference on Preventing Preterm Birth and the Millennium Development Goals (Goal 4, Reduce Child Mortality). Spontaneous preterm birth (SPTB) accounts for 70-80% of preterm births, with the remainder being medically induced. Bacterial vaginosis (BV) is a common condition that has consistently been associated with SPTB. Many bacteria associated with BV remain difficult to detect using cultivation methods, but are easily detected using molecular methods. Despite the consistent association between BV and SPTB, treating BV during pregnancy has failed to reduce SPTB in numerous clinical trials, raising critical questions: Do key bacterial species mediate the risk of SPTB? Is treatment of BV during pregnancy too late to reduce SPTB? Our multidisciplinary team has been at the forefront in developing cutting-edge laboratory techniques and applying them in carefully conducted epidemiological studies to make important discoveries linking disruption of the vaginal microbiota to adverse reproductive health outcomes. In this proposal, we will test the overarching hypothesis that high-risk vaginal bacterial species present near the time of conception colonize the uterine cavity, causing sub-clinical inflammation, and leading to SPTB. We will leverage access to facilities, highly trained staff, and a population of Kenyan women attending preconception clinics supported with US CDC and NIH funding, to make this unique case-cohort study possible. Aim 1 will employ broad-range 16S ribosomal RNA gene polymerase chain reaction (PCR) and deep sequencing to compare vaginal bacterial community structure, diversity, richness, and the presence of key species near conception, in women with SPTB vs. term delivery. Aim 2 will use highly sensitive quantitative PCR (qPCR) assays to compare the presence and concentrations of key vaginal bacterial species near conception in women with SPTB vs. term birth, validating or refuting preliminary associations observed in Aim 1. Aim 3 will utilize fetal membrane and umbilical cord samples collected at delivery, combining species-specific qPCR assays and histological examination, to determine whether vaginal bacteria associated with SPTB in Aims 1 and 2 ascend to the upper genital tract and cause inflammation. In parallel, we will examine associations between key vaginal bacterial species during pregnancy and SPTB, allowing us to directly compare the risk of SPTB associated with vaginal bacteria identified in the peri-conception period vs. during pregnancy. Identification of strong relationships between bacterial species and communities present close to the time of conception and SPTB is expected to shift our paradigm for understanding how vaginal microbiota influences women's risk of SPTB. These data could inform development of targeted interventions based on elimination of high-risk species and promotion of low-risk vaginal bacterial communities in women planning a pregnancy, particularly after prior SPTB.